Donkey Anti-Rabbit IgG/PE

The Donkey Anti-Rabbit IgG/PE antibody is a versatile reagent for various applications, including Immunofluorescence (IF) and Flow Cytometry (FCM). Below are the protocols for each validated application.

For IF, the following steps are recommended:

- Reagents or Buffers: Phosphate-buffered saline (PBS), 4% paraformaldehyde for fixation, and a mounting medium with DAPI for nuclear staining.

- Sample Preparation: Fix cells or tissue sections with 4% paraformaldehyde, permeabilize with 0.1% Triton X-100 if necessary, and block with 5% normal donkey serum.

- Antibody Incubation: Incubate the sample with the primary rabbit antibody, followed by the Donkey Anti-Rabbit IgG/PE at a dilution of 1:100-1000 in PBS containing 1% bovine serum albumin (BSA) for 1 hour at room temperature.

- Washing or Detection: Wash the sample thoroughly with PBS, and then mount with a DAPI-containing mounting medium for nuclear counterstaining.

- Technical Notes: Optimize the primary antibody concentration and the Donkey Anti-Rabbit IgG/PE dilution for the best signal-to-noise ratio.

For FCM, the following steps are recommended:

- Reagents or Buffers: PBS, 1% BSA, and a fixation buffer if necessary.

- Sample Preparation: Prepare cell suspensions and block with 5% normal donkey serum if necessary.

- Antibody Incubation: Incubate cells with the primary rabbit antibody, followed by the Donkey Anti-Rabbit IgG/PE at a dilution of 1:100-1000 in PBS containing 1% BSA for 30 minutes to 1 hour on ice.

- Washing or Detection: Wash cells with PBS, and then analyze by flow cytometry.

- Technical Notes: Use appropriate controls, such as isotype controls for the primary antibody and a secondary antibody-only control, to validate the specificity of the staining.

Both IF and FCM require optimization of the antibody dilutions and conditions to achieve the best results. Always use proper controls to validate the specificity of the staining.

Use assay conditions validated for IF, apply Donkey Anti-Rabbit IgG/PE at the recommended working dilution, include proper positive and negative controls, perform thorough wash steps, and document incubation time, temperature, and detection conditions for reproducible performance. Recommended dilution: FCM=1:100-1000;

IF=1:100-1000.

Use assay conditions validated for FCM, apply Donkey Anti-Rabbit IgG/PE at the recommended working dilution, include proper positive and negative controls, perform thorough wash steps, and document incubation time, temperature, and detection conditions for reproducible performance. Recommended dilution: FCM=1:100-1000;

IF=1:100-1000.